Fibrinogen-binding and fibronectin-binding assay.

<p>The amount of sortase A-anchored, LPXTG-motif containing fibrinogen-binding protein (A) and fibronectin-binding protein (B) was measured by flow cytometry using FITC-conjugated fibrinogen and HiLyte Fluor ^TM 488-conjugated fibronectin respectively. The percentage binding was determined relative to highest value obtained for that experiment. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001). <i>(Since the Newman isolate has truncations in both FnBP-A and FnBP-B</i>, <i>it was not tested in the fibronectin binding assay)</i>.</p

Molecular basis of surface anchored protein A deficiency in the <i>Staphylococcus aureus</i> strain Wood 46 - Fig 3

<p><b>(A) Amino acid composition of sortase A.</b> The amino acid sequence of sortase A in Cowan 1, Seattle 1945, Newman and Wood 46 were aligned. Amino acids from position 155 to the end of the protein are shown. Wood 46 contains a 12bp deletion at the 3’ end of the <i>srtA</i> gene that leads to a corresponding loss of four amino acids at the C-terminus of the protein. <b>(B). Promotor and upstream regulatory region of <i>srtA</i> in Wood 46</b>. Consensus promoter and upstream regulatory sequence from Cowan 1, Seattle 1945 and Newman was aligned with that of Wood 46. The boxes highlight regions with mutations. The transcription start site ATG is indicated as +1.</p

Cell wall-associated protein A.

<p>Protein A expressed on the surface of <i>S</i>. <i>aureus</i> strains Cowan1, Seattle 1945 (25923), Wood 46, Newman WT and its corresponding deletion mutants, <i>Δ</i>sbi, <i>Δ</i>spa and <i>Δ</i>sbi-<i>Δ</i>spa, as measured by flow cytometry with chicken anti-protein A antibody. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001).</p

Expression levels of <i>spa</i> and <i>srtA</i>.

<p>Quantitative real-time PCR was used to measure the amount of (A) <i>spa</i> mRNA and (B) <i>srtA</i> mRNA in log phase bacterial cells. Relative gene expression was calculated using the ΔΔC_T method and expressed as fold change. 16S rRNA was used as the endogenous control. The amount of <i>srtA</i> mRNA in Wood 46 was below the detection limit of the assay. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001). ND- not detected.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth

The marine archaeon <i>Nanoarchaeum equitans</i> is dependent on direct physical contact with its host, the hyperthermophile <i>Ignicoccus hospitalis</i>. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted <i>I. hospitalis-N. equitans</i> proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of <i>Caldicellulosiruptor obsidiansis</i> cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When <i>C. obsidiansis</i> was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation

Defining the Boundaries and Characterizing the Landscape of Functional Genome Expression in Vascular Tissues of <i>Populus</i> using Shotgun Proteomics

Current state-of-the-art experimental and computational proteomic approaches were integrated to obtain a comprehensive protein profile of <i>Populus</i> vascular tissue. This featured: (1) a large sample set consisting of two genotypes grown under normal and tension stress conditions, (2) bioinformatics clustering to effectively handle gene duplication, and (3) an informatics approach to track and identify single amino acid polymorphisms (SAAPs). By applying a clustering algorithm to the <i>Populus</i> database, the number of protein entries decreased from 64689 <i>proteins</i> to a total of 43069 <i>protein groups</i>, thereby reducing 7505 identified proteins to a total of 4226 protein groups, in which 2016 were singletons. This reduction implies that ∼50% of the measured proteins shared extensive sequence homology. Using conservative search criteria, we were able to identify 1354 peptides containing a SAAP and 201 peptides that become tryptic due to a K or R substitution. These newly identified peptides correspond to 502 proteins, including 97 previously unidentified proteins. In total, the integration of deep proteome measurements on an extensive sample set with protein clustering and peptide sequence variants provided an exceptional level of proteome characterization for <i>Populus</i>, allowing us to spatially resolve the vascular tissue proteome